Characterization of a Lichenase Isolated from Soil Metagenome

Sang-Yoon  Kim; Doo-Byoung  Oh; Ohsuk  Kwon

doi:10.4014/jmb.1406.06012

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Research article

Environmental Microbiology and Engineering

Characterization of a Lichenase Isolated from Soil Metagenome

Sang-Yoon Kim ¹, Doo-Byoung Oh ^{1, 2} and Ohsuk Kwon ^{1, 2*}

¹Synthetic Biology and Bioengineering, Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Republic of Korea, ²Biosystems and Bioengineering Program, School of Science, University of Science and Technology, Daejeon 305-350, Republic of Korea

Received: June 5, 2014; Accepted: August 13, 2014

J. Microbiol. Biotechnol. 2014; 24(12): 1699-1706

Published December 28, 2014 https://doi.org/10.4014/jmb.1406.06012

Abstract

A lichenase gene (mt-lic) was identified for the first time through function-based screening of a soil metagenomic library. Its deduced amino acid sequence exhibited a high degree of homology with endo-β-1,3-1,4-glucanase (having both lichenase and chitosanase activities), encoded by the bgc gene of Bacillus circulans WL-12. The recombinant lichenase overexpressed and purified from Escherichia coli was able to efficiently hydrolyze both barley β-glucan and lichenan. The enzyme showed maximal activity at a pH of 6.0 at 50°C, with Azo-barley-glucan as the substrate. The metal ions Mn2+, Mg2+, Ca2+, and Fe2+ enhanced the enzymatic activity, whereas the Cu2+ and Zn2+ ions inhibited the enzymatic activity. The Km and Vmax values of the purified lichenase were determined to be 0.45 mg/ml and 24.83 U/min/mg of protein, respectively.

Keywords

Endo-β-1,3-1,4-glucanase, Glycosyl hydrolase family 8, Lichenase, Recombinant protein, Soil metagenome

References

Adachi W, Sakihama Y, Shimizu S, Sunami T, Fukazawa T, Suzuki M, et al. 2004. Crystal structure of family GH-8 chitosanase with subclass II specificity from Bacillus sp. K17. J. Mol. Biol. 343: 785-795.
Akita M, Kayatama K, Hatada Y, Ito S, Horikoshi K. 2005. A novel β-glucanase gene from Bacillus halodurans C-125. FEMS Microbiol. Lett. 248: 9-15.
Anderson M A, S tone B A. 1 975. A n ew s ubstrate for investigating the specificity of β-glucan hydrolases. FEBS Lett. 52: 202-207.
Apiraksakorn J, Nitisinprasert S, Levin RE. 2008. Grass degrading β-1,3-1,4-D-glucanases from Bacillus subtilis GN156:purification and characterization of glucanase J1 and pJ2 possessing extremely acidic pI. Appl. Biochem. Biotechnol. 149: 53-66.
Beckmann L, Simon O, Vahjen W. 2006. Isolation and identification of mixed linked β-glucan degrading bacteria in the intestine of broiler chickens and partial characterization of respective 1,3-1,4-β-glucanase activities. J. Basic Microbiol. 46: 175-185.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.
Bueno A, Vazquez de Aldana CR, Correa J, Villa TG, del Rey F. 1990. Synthesis and secretion of a Bacillus circulans WL-12 1,3-1,4-β-D-glucanase in Escherichia coli. J. Bacteriol. 172: 2160-2167.
Buliga GS, Brant DA, Fincher GB. 1986. The sequence statistics and solution conformation of a barley (1-3, 1-4)-βD-glucan. Carbohydr. Res. 157: 139-156.
Chaari F, Bhiri F, Blibech M, Maktouf S, Ellouz-Chaabouni S, Ellouz-Ghorbel R. 2012. Potential application of two thermostable lichenases from a newly isolated Bacillus licheniformis UEB CF: Purification and characterization. Process Biochem. 47:509-516.
Daniel R. 2005. The metagenomics of soil. Nat. Rev. Microbiol. 3: 470-478.
Feng Y, Duan CJ, Pang H, Mo XC, Wu CF, Yu Y, et al. 2007. Cloning and identification of novel cellulase genes from uncultured microorganisms in rabbit cecum and characterization of the expressed cellulases. Appl. Microbiol. Biotechnol. 75:319-328.
Fernandez-Arrojo L, Guazzaroni ME, Lopez-Cortes N, Beloqui A, Ferrer M. 2010. Metagenomic era for biocatalyst identification. Curr. Opin. Biotechnol. 21: 725-733.
Hakamada Y, Endo K, Takizawa S, Kobayashi T, Shirai T, Yamane T, Ito S. 2002. Enzymatic properties, crystallization, and deduced amino acid sequence of an alkaline endoglucanase from Bacillus circulans. Biochim. Biophys. Acta 1570: 174-180.
Handelsman J. 2004. Metagenomics: application of genomics to uncultured microorganisms. Microbiol. Mol. Biol. Rev. 68:669-685.
Hong MR, Kim YS, Joo AR, Lee JK, Kim YS, Oh DK. 2009. Purification and characterization of a thermostable β-1,3-1,4glucanase from Laetiporus sulphureus var. miniatus. J. Microbiol. Biotechnol. 19: 818-822.
Jiang C, Li S X, Luo FF, Jin K, Wang Q, Hao ZY, et al. 2011. Biochemical characterization of two novel β-glucosidase genes by metagenome expression cloning. Bioresour. Technol. 102:3272-3278.
Kim D , Kim SN, B aik KS , Park SC, L im CH, K im JO, et al. 2011. Screening and characterization of a cellulase gene from the gut microflora of abalone using metagenomic library. J. Microbiol. 49: 141-145.
Kim JY. 2003. Overproduction and secretion of Bacillus circulans endo-b-1,3-1,4-glucanase gene (bglBC1) in B. subtilis and B. megaterium. Biotechnol. Lett. 25: 1445-1449.
Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.
Lloberas J, Querol E, Bernués J. 1988. Purification and characterization of endo-β-1,3-1,4-d-glucanase activity from Bacillus licheniformis. Appl. Microbiol. Biotechnol. 29: 32-38.
Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS. 2002. Microbial cellulose utilization: fundamentals and biotechnology. Microbiol. Mol. Biol. Rev. 66: 506-577.
Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426-428.
Miroux B, Walker JE. 1996. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260: 289-298.
Mitsutomi M, Isono M, Uchiyama A, Nikaidou N, Ikegami T, Watanabe T. 1998. Chitosanase activity of the enzyme previously reported as β-1,3-1,4-glucanase from Bacillus circulans WL-12. Biosci. Biotechnol. Biochem. 62: 2107-2114.
Muller JJ, Thomsen KK, Heinemann U. 1998. Crystal structure of barley 1,3-1,4-β-glucanase at 2.0-Å resolution and comparison with Bacillus 1,3-1,4-β-glucanase. J. Biol. Chem. 273: 3438-3446.
Notredame C, Higgins DG, Heringa J. 2000. T-Coffee: A novel method for fast and accurate multiple sequence alignment. J. Mol. Biol. 302: 205-217.
Olsen O, Borriss R, Simon O, Thomsen KK. 1991. Hybrid Bacillus (1-3,1-4)-β-glucanases: engineering thermostable enzymes by construction of hybrid genes. Mol. Gen. Genet. 225: 177-185.
Pang H, Zhang P, Duan CJ, Mo XC, Tang JL, Feng JX. 2009. Identification of cellulase genes from the metagenomes of compost soils and functional characterization of one novel endoglucanase. Curr. Microbiol. 58: 404-408.
Petersen TN, Brunak S, von Heijne G, Nielsen H. 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8: 785-786.
Planas A. 2000. Bacterial 1,3-1,4-β-glucanases: structure, function and protein engineering. Biochim. Biophys. Acta 1543: 361-382.
Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C, et al. 2012. The Pfam protein families database. Nucleic Acids Res. 40: D290-D301.
Schimming S, Schwarz WH, Staudenbauer WL. 1991. Properties of a thermoactive β-1,3-1,4-glucanase (lichenase) from Clostridium thermocellum expressed in Escherichia coli. Biochem. Biophys. Res. Commun. 177: 447-452.
Shinoda S, Kanamasa S, Arai M. 2012. Cloning of an endoglycanase gene from Paenibacillus cookii and characterization of the recombinant enzyme. Biotechnol. Lett. 34: 281-286.
Teng D, Wang JH, Fan Y, Yang YL, Tian ZG, Luo J, et al. 2006. Cloning of β-1,3-1,4-glucanase gene from Bacillus licheniformis EGW039 (CGMCC 0635) and its expressiona in Escherichia coli BL21 (DE3). Appl. Microbiol. Biotechnol. 72:705-712.
Walter J, Mangold M, Tannock GW. 2005. Construction, analysis, and β-glucanase screening of a bacterial artificial chromosome library from the large-bowel microbiota of mice. Appl. Environ. Microbiol. 71: 2347-2354.
Yoo DH, Lee BH, Chang PS, Lee HG, Yoo SH. 2007. Improved quantitative analysis of oligosaccharides from lichenase-hydrolyzed water-soluble barley β-glucans by highperformance anion-exchange chromatography. J. Agric. Food Chem. 55: 1656-1662.

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Article

Research article

J. Microbiol. Biotechnol. 2014; 24(12): 1699-1706

Published online December 28, 2014 https://doi.org/10.4014/jmb.1406.06012

Characterization of a Lichenase Isolated from Soil Metagenome

Sang-Yoon Kim ¹, Doo-Byoung Oh ^{1, 2} and Ohsuk Kwon ^{1, 2*}

¹Synthetic Biology and Bioengineering, Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Republic of Korea, ²Biosystems and Bioengineering Program, School of Science, University of Science and Technology, Daejeon 305-350, Republic of Korea

Received: June 5, 2014; Accepted: August 13, 2014

Abstract

A lichenase gene (mt-lic) was identified for the first time through function-based screening of a
soil metagenomic library. Its deduced amino acid sequence exhibited a high degree of
homology with endo-β-1,3-1,4-glucanase (having both lichenase and chitosanase activities),
encoded by the bgc gene of Bacillus circulans WL-12. The recombinant lichenase overexpressed
and purified from Escherichia coli was able to efficiently hydrolyze both barley β-glucan and
lichenan. The enzyme showed maximal activity at a pH of 6.0 at 50°C, with Azo-barley-glucan
as the substrate. The metal ions Mn2+, Mg2+, Ca2+, and Fe2+ enhanced the enzymatic activity,
whereas the Cu2+ and Zn2+ ions inhibited the enzymatic activity. The Km and Vmax values of the
purified lichenase were determined to be 0.45 mg/ml and 24.83 U/min/mg of protein,
respectively.

Keywords: Endo-β-1,3-1,4-glucanase, Glycosyl hydrolase family 8, Lichenase, Recombinant protein, Soil metagenome

Abstract

References

Adachi W, Sakihama Y, Shimizu S, Sunami T, Fukazawa T, Suzuki M, et al. 2004. Crystal structure of family GH-8 chitosanase with subclass II specificity from Bacillus sp. K17. J. Mol. Biol. 343: 785-795.
Akita M, Kayatama K, Hatada Y, Ito S, Horikoshi K. 2005. A novel β-glucanase gene from Bacillus halodurans C-125. FEMS Microbiol. Lett. 248: 9-15.
Anderson M A, S tone B A. 1 975. A n ew s ubstrate for investigating the specificity of β-glucan hydrolases. FEBS Lett. 52: 202-207.
Apiraksakorn J, Nitisinprasert S, Levin RE. 2008. Grass degrading β-1,3-1,4-D-glucanases from Bacillus subtilis GN156:purification and characterization of glucanase J1 and pJ2 possessing extremely acidic pI. Appl. Biochem. Biotechnol. 149: 53-66.
Beckmann L, Simon O, Vahjen W. 2006. Isolation and identification of mixed linked β-glucan degrading bacteria in the intestine of broiler chickens and partial characterization of respective 1,3-1,4-β-glucanase activities. J. Basic Microbiol. 46: 175-185.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.
Bueno A, Vazquez de Aldana CR, Correa J, Villa TG, del Rey F. 1990. Synthesis and secretion of a Bacillus circulans WL-12 1,3-1,4-β-D-glucanase in Escherichia coli. J. Bacteriol. 172: 2160-2167.
Buliga GS, Brant DA, Fincher GB. 1986. The sequence statistics and solution conformation of a barley (1-3, 1-4)-βD-glucan. Carbohydr. Res. 157: 139-156.
Chaari F, Bhiri F, Blibech M, Maktouf S, Ellouz-Chaabouni S, Ellouz-Ghorbel R. 2012. Potential application of two thermostable lichenases from a newly isolated Bacillus licheniformis UEB CF: Purification and characterization. Process Biochem. 47:509-516.
Daniel R. 2005. The metagenomics of soil. Nat. Rev. Microbiol. 3: 470-478.
Feng Y, Duan CJ, Pang H, Mo XC, Wu CF, Yu Y, et al. 2007. Cloning and identification of novel cellulase genes from uncultured microorganisms in rabbit cecum and characterization of the expressed cellulases. Appl. Microbiol. Biotechnol. 75:319-328.
Fernandez-Arrojo L, Guazzaroni ME, Lopez-Cortes N, Beloqui A, Ferrer M. 2010. Metagenomic era for biocatalyst identification. Curr. Opin. Biotechnol. 21: 725-733.
Hakamada Y, Endo K, Takizawa S, Kobayashi T, Shirai T, Yamane T, Ito S. 2002. Enzymatic properties, crystallization, and deduced amino acid sequence of an alkaline endoglucanase from Bacillus circulans. Biochim. Biophys. Acta 1570: 174-180.
Handelsman J. 2004. Metagenomics: application of genomics to uncultured microorganisms. Microbiol. Mol. Biol. Rev. 68:669-685.
Hong MR, Kim YS, Joo AR, Lee JK, Kim YS, Oh DK. 2009. Purification and characterization of a thermostable β-1,3-1,4glucanase from Laetiporus sulphureus var. miniatus. J. Microbiol. Biotechnol. 19: 818-822.
Jiang C, Li S X, Luo FF, Jin K, Wang Q, Hao ZY, et al. 2011. Biochemical characterization of two novel β-glucosidase genes by metagenome expression cloning. Bioresour. Technol. 102:3272-3278.
Kim D , Kim SN, B aik KS , Park SC, L im CH, K im JO, et al. 2011. Screening and characterization of a cellulase gene from the gut microflora of abalone using metagenomic library. J. Microbiol. 49: 141-145.
Kim JY. 2003. Overproduction and secretion of Bacillus circulans endo-b-1,3-1,4-glucanase gene (bglBC1) in B. subtilis and B. megaterium. Biotechnol. Lett. 25: 1445-1449.
Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.
Lloberas J, Querol E, Bernués J. 1988. Purification and characterization of endo-β-1,3-1,4-d-glucanase activity from Bacillus licheniformis. Appl. Microbiol. Biotechnol. 29: 32-38.
Lynd LR, Weimer PJ, van Zyl WH, Pretorius IS. 2002. Microbial cellulose utilization: fundamentals and biotechnology. Microbiol. Mol. Biol. Rev. 66: 506-577.
Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426-428.
Miroux B, Walker JE. 1996. Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260: 289-298.
Mitsutomi M, Isono M, Uchiyama A, Nikaidou N, Ikegami T, Watanabe T. 1998. Chitosanase activity of the enzyme previously reported as β-1,3-1,4-glucanase from Bacillus circulans WL-12. Biosci. Biotechnol. Biochem. 62: 2107-2114.
Muller JJ, Thomsen KK, Heinemann U. 1998. Crystal structure of barley 1,3-1,4-β-glucanase at 2.0-Å resolution and comparison with Bacillus 1,3-1,4-β-glucanase. J. Biol. Chem. 273: 3438-3446.
Notredame C, Higgins DG, Heringa J. 2000. T-Coffee: A novel method for fast and accurate multiple sequence alignment. J. Mol. Biol. 302: 205-217.
Olsen O, Borriss R, Simon O, Thomsen KK. 1991. Hybrid Bacillus (1-3,1-4)-β-glucanases: engineering thermostable enzymes by construction of hybrid genes. Mol. Gen. Genet. 225: 177-185.
Pang H, Zhang P, Duan CJ, Mo XC, Tang JL, Feng JX. 2009. Identification of cellulase genes from the metagenomes of compost soils and functional characterization of one novel endoglucanase. Curr. Microbiol. 58: 404-408.
Petersen TN, Brunak S, von Heijne G, Nielsen H. 2011. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat. Methods 8: 785-786.
Planas A. 2000. Bacterial 1,3-1,4-β-glucanases: structure, function and protein engineering. Biochim. Biophys. Acta 1543: 361-382.
Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C, et al. 2012. The Pfam protein families database. Nucleic Acids Res. 40: D290-D301.
Schimming S, Schwarz WH, Staudenbauer WL. 1991. Properties of a thermoactive β-1,3-1,4-glucanase (lichenase) from Clostridium thermocellum expressed in Escherichia coli. Biochem. Biophys. Res. Commun. 177: 447-452.
Shinoda S, Kanamasa S, Arai M. 2012. Cloning of an endoglycanase gene from Paenibacillus cookii and characterization of the recombinant enzyme. Biotechnol. Lett. 34: 281-286.
Teng D, Wang JH, Fan Y, Yang YL, Tian ZG, Luo J, et al. 2006. Cloning of β-1,3-1,4-glucanase gene from Bacillus licheniformis EGW039 (CGMCC 0635) and its expressiona in Escherichia coli BL21 (DE3). Appl. Microbiol. Biotechnol. 72:705-712.
Walter J, Mangold M, Tannock GW. 2005. Construction, analysis, and β-glucanase screening of a bacterial artificial chromosome library from the large-bowel microbiota of mice. Appl. Environ. Microbiol. 71: 2347-2354.
Yoo DH, Lee BH, Chang PS, Lee HG, Yoo SH. 2007. Improved quantitative analysis of oligosaccharides from lichenase-hydrolyzed water-soluble barley β-glucans by highperformance anion-exchange chromatography. J. Agric. Food Chem. 55: 1656-1662.